Viral envelope glycoprotein processing by proprotein convertases.

The proprotein convertases (PCs) are a family of nine mammalian enzymes that play key roles in the maintenance of cell homeostasis by activating or inactivating proteins via limited proteolysis under temporal and spatial control. A wide range of pathogens, including major human pathogenic viruses can hijack cellular PCs for their own purposes. In particular, productive infection with many enveloped viruses critically depends on the processing of their fusion-active viral envelope glycoproteins by cellular PCs. Based on their crucial role in virus-host interaction, PCs can be important determinants for viral pathogenesis and represent promising targets of therapeutic antiviral intervention. In the present review we will cover basic aspects and recent developments of PC-mediated maturation of viral envelope glycoproteins of selected medically important viruses. The molecular mechanisms underlying the recognition of PCs by viral glycoproteins will be described, including recent findings demonstrating differential PC-recognition of viral and cellular substrates. We will further discuss a possible scenario how viruses during co-evolution with their hosts adapted their glycoproteins to modulate the activity of cellular PCs for their own benefit and discuss the consequences for virus-host interaction and pathogenesis. Particular attention will be given to past and current efforts to evaluate cellular PCs as targets for antiviral therapeutic intervention, with emphasis on emerging highly pathogenic viruses for which no efficacious drugs or vaccines are currently available.

Validation and long-term evaluation of a modified on-line chiral analytical method for therapeutic drug monitoring of (R,S)-methadone in clinical samples.

Matrix effects, which represent an important issue in liquid chromatography coupled to mass spectrometry or tandem mass spectrometry detection, should be closely assessed during method development. In the case of quantitative analysis, the use of stable isotope-labelled internal standard with physico-chemical properties and ionization behaviour similar to the analyte is recommended. In this paper, an example of the choice of a co-eluting deuterated internal standard to compensate for short-term and long-term matrix effect in the case of chiral (R,S)-methadone plasma quantification is reported. The method was fully validated over a concentration range of 5-800 ng/mL for each methadone enantiomer with satisfactory relative bias (-1.0 to 1.0%), repeatability (0.9-4.9%) and intermediate precision (1.4-12.0%). From the results obtained during validation, a control chart process during 52 series of routine analysis was established using both intermediate precision standard deviation and FDA acceptance criteria. The results of routine quality control samples were generally included in the +/-15% variability around the target value and mainly in the two standard deviation interval illustrating the long-term stability of the method. The intermediate precision variability estimated in method validation was found to be coherent with the routine use of the method. During this period, 257 trough concentration and 54 peak concentration plasma samples of patients undergoing (R,S)-methadone treatment were successfully analysed for routine therapeutic drug monitoring.

Comparison of porcine and human coagulation by thrombelastometry.

Introduction: Although the pig is a standard model for the evaluation of various diseases in humans, including coagulopathy, it is not clear whether results in animals can be extrapolated to man.Materials and methods: In 75 anesthetized pigs, we assessed reagent-supported thrombelastometry (ExTEM (R)), platelet-blocked thrombelastometry (FibTEM (R)), and aprotinin thrombelastometry (ApTEM (R)). Results were compared to values from 13 anesthetized humans.Results (median, 95% CI): ExTEM (R) : While clot strength was comparable in pigs (66 mm, 65-67 mm) and in humans (64 mm, 60-68 mm; NS), clotting time in animals was longer (pigs 64 s, 62-66 s; humans 55 s, 49-71 s; P<0.05) and clot formation time shorter (pigs 52 s, 49-54 s; humans 83 s, 67-98 s, P<0.001). The clot lysis index at 30 minutes was lower in animals (96.9%, 95.1-97.3%) than in humans (99.5%, 98.6-99.9%; P<0.001). ApTEM (R) showed no hyperfibrinolysis in animals. Modification of the anesthesia protocol in animals resulted in significant ExTEM (R) changes. FibTEM (R) : Complete platelet inhibition yielded significantly higher platelet contribution to clot strength in pigs (79%, 76-81%) than in humans (73%, 71-77%; P<0.05), whereas fibrinogen contribution to clot strength was higher in humans (27%, 24-29%) than in animals (21%, 19-24%; P<0.05).Conclusions: Maximum clot firmness is comparable in human and porcine blood. However, clot lysis, platelet and fibrinogen contribution to clot strength, as well as initiation and propagation of clotting, are considerably different between pigs and humans. In addition, anesthesic drugs seem to influence thrombelastometry in animals. Accordingly, coagulation abnormalities in pigs subjected to diseases may not necessarily represent the coagulation profile in sick patients. (C) 2011 Elsevier Ltd. All rights reserved.

Prevalence and clinical importance of mesenteric venous thrombosis in the Swiss Inflammatory Bowel Disease Cohort

OBJECTIVE: The purpose of this study was to evaluate the prevalence of mesenteric venous thrombosis (MVT) in the Swiss Inflammatory Bowel Disease Cohort Study and to correlate MVT with clinical outcome. MATERIALS AND METHODS: Abdominal portal phase CT was used to examine patients with inflammatory bowel disease (IBD). Two experienced abdominal radiologists retrospectively analyzed the images, focusing on the superior and inferior mesenteric vein branches and looking for signs of acute or chronic thrombosis. The location of abnormalities was registered. The presence of MVT was correlated with IBD-related radiologic signs and complications. RESULTS: The cases of 160 patients with IBD (89 women, 71 men; Crohn disease [CD], 121 patients; ulcerative colitis [UC], 39 patients; median age at diagnosis, 27 years for patients with CD, 32 years for patients with UC) were analyzed. MVT was detected in 43 patients with IBD (26.8%). One of these patients had acute MVT; 38, chronic MVT; and four, both. The prevalence of MVT did not differ between CD (35/121 [28.9%]) and UC (8/39 [20.5%]) (p = 0.303). The location of thrombosis was different between CD and UC (CD, jejunal or ileal veins only [p = 0.005]; UC, rectocolic veins only [p = 0.001]). Almost all (41/43) cases of thrombosis were peripheral. MVT in CD patients was more frequently associated with bowel wall thickening (p = 0.013), mesenteric fat hypertrophy (p = 0.005), ascites (p = 0.002), and mesenteric lymph node enlargement (p = 0.036) and was associated with higher rate of bowel stenosis (p < 0.001) and more intestinal IBD-related surgery (p = 0.016) in the outcome. Statistical analyses for patients with UC were not relevant because of the limited population (n = 8). CONCLUSION: MVT is frequently found in patients with IBD. Among patients with CD, MVT is associated with bowel stenosis and CD-related intestinal surgery.

Acute aphasia after right hemisphere stroke

Abstract Right hemispheric stroke aphasia (RHSA) rarely occurs in right- or left-handed patients with their language representation in right hemisphere (RH). For right-handers, the term crossed aphasia is used. Single cases, multiple cases reports, and reviews suggest more variable anatomo-clinical correlations. We included retrospectively from our stroke data bank 16 patients (right- and left-handed, and ambidextrous) with aphasia after a single first-ever ischemic RH stroke. A control group was composed of 25 successive patients with left hemispheric stroke and aphasia (LHSA). For each patient, we analyzed four modalities of language (spontaneous fluency, naming, repetition, and comprehension) and recorded eventual impairment: (1) on admission (hyperacute) and (2) between day 3 and 14 (acute). Lesion volume and location as measured on computed tomography (CT) and magnetic resonance imaging (MRI) were transformed into Talairach stereotaxic space. Nonparametric statistics were used to compare impaired/nonimpaired patients. Comprehension and repetition were less frequently impaired after RHSA (respectively, 56% and 50%) than after LHSA (respectively, 84% and 80%, P = 0.05 and 0.04) only at hyperacute phase. Among RHSA, fewer left-handers/ambidextrous than right-handers had comprehension disorders at second evaluation (P = 0.013). Mean infarct size was similar in RHSA and LHSA with less posterior RHSA lesions (caudal to the posterior commissure). Comprehension and repetition impairments were more often associated with anterior lesions in RHSA (Fisher's exact test, P < 0.05). Despite the small size of the cohort, our findings suggest increased atypical anatomo-functional correlations of RH language representation, particularly in non-right-handed patients. Rapport de synthèse : Des aphasies secondaires à un accident vasculaire ischémique cérébral (AVC) hémisphérique droit sont rarement rencontrées chez des patients droitiers ou gauchers avec une représentation du langage dans l'hémisphère droit. Chez les droitiers, on parle d'aphasie croisée. Plusieurs études sur le sujet ont suggéré des corrélations anatomocliniques plus variables. Dans notre étude, nous avons inclus rétrospectivement, à partir d'une base de données de patients avec un AVC, seize patients (droitiers, gauchers et ambidextres) souffrant d'une aphasie suite à un premier et unique AVC ischémique hémisphérique droit. Un groupe contrôle est composé de vingt-cinq patients successifs avec une aphasie suite à un AVC ischémique hémisphérique gauche. Pour chaque patient, nous avons analysé quatre modalités de langage, à savoir la fluence spontanée, la dénomination, la répétition et la compréhension et leur éventuelle atteinte à deux moments distincts : 1) à l'admission (phase hyperaiguë) et 2) entre le 3e et le 14e jour (phase aiguë). Le volume et la localisation de la lésion mesurés, soit sur un CT-scanner soit sur une imagerie par résonance magnétique cérébrale, ont été analysés à l'aide de l'échelle stéréotaxique de Talairach. Des statistiques non paramétriques ont été utilisées pour comparer les patients atteints et non atteints. . La compréhension et la répétition étaient moins souvent atteintes, seulement en phase hyperaiguë, après une aphasie suite à un AVC hémisphérique droit (resp. 56% et 50%) plutôt que gauche (resp. 84 % et 80%, p= 0.05 et 0.04). Parmi les aphasies suite à un AVC ischémique hémisphérique droit, moins de gauchers et d'ambidextres que de droitiers avaient des troubles de la compréhension lors de la seconde évaluation (p=0.013}. La .taille moyenne de la zone infarcie était semblable entre les aphasies droites et gauches, avec moins de lésions postérieures (caudale à la commissure postérieure) lors des aphasies droites. Les troubles de la répétition et de la compréhension étaient plus souvent associés à des lésions antérieures lors d'aphasie droite. (Fischer's exact test, p>0.05). Malgré la petite taille de notre cohorte de patients, ces résultats suggèrent une augmentation des corrélations anatomocliniques atypiques lors d'une représentation du langage dans l'hémisphère droit, surtout chez les patients non droitiers.

Particulate formulations for the delivery of poly(I:C) as vaccine adjuvant.

Current research and development of antigens for vaccination often center on purified recombinant proteins, viral subunits, synthetic oligopeptides or oligosaccharides, most of them suffering from being poorly immunogenic and subject to degradation. Hence, they call for efficient delivery systems and potent immunostimulants, jointly denoted as adjuvants. Particulate delivery systems like emulsions, liposomes, nanoparticles and microspheres may provide protection from degradation and facilitate the co-formulation of both the antigen and the immunostimulant. Synthetic double-stranded (ds) RNA, such as polyriboinosinic acid-polyribocytidylic acid, poly(I:C), is a mimic of viral dsRNA and, as such, a promising immunostimulant candidate for vaccines directed against intracellular pathogens. Poly(I:C) signaling is primarily dependent on Toll-like receptor 3 (TLR3), and on melanoma differentiation-associated gene-5 (MDA-5), and strongly drives cell-mediated immunity and a potent type I interferon response. However, stability and toxicity issues so far prevented the clinical application of dsRNAs as they undergo rapid enzymatic degradation and bear the potential to trigger undue immune stimulation as well as autoimmune disorders. This review addresses these concerns and suggests strategies to improve the safety and efficacy of immunostimulatory dsRNA formulations. The focus is on technological means required to lower the necessary dosage of poly(I:C), to target surface-modified microspheres passively or actively to antigen-presenting cells (APCs), to control their interaction with non-professional phagocytes and to modulate the resulting cytokine secretion profile.

Long term outcome after mitral valve repair : a risk factor analysis

Objectif: La réparation de la valve mitrale constitue le traitement de choix pour restaurer ta fonction de celle-ci. Elle est actuellement reconnue pour garantir une bonne évolution à long terme. Dans le but de faciliter les décisions périopératoires, nous avons analysé nos patients afin de déterminer les facteurs de risque ayant affecté leur évolution. Méthodes: Nous avons étudié rétrospectivement 175 premiers patients consécutifs (âge moyen : 64 +/-10.4 ans ;113 hommes) qui ont subi une réparation primaire de la valve mitrale associée à toute autre intervention cardiaque entre 1986 et 1998. Les facteurs de risque influençant le taux de réopération et la survie à long terme ont été analysés de manière uni et multivariée. Résultats: La mortalité opératoire était de 3.4 % (6 décès, 0 -22 et jours post-opératoires). La mortalité tardive était de 9.1 % (16 décès, 3e-125e mois post-opératoires). Cinq patients ont dû être réopérés. L'analyse actuarielle selon Kaplan-Meier a montré une survie à 1 année de 96 +l-1 %, une survie à 5 ans de 88 +/- 3 % et une survie à 10 ans de 69 +/- 8 %. Après 1 année, la fraction de population sans réopération était de 99 %, elle était de 97 +/-2 % après 5 ans et de 88+/-6 % après 10 ans. L'analyse multivariée a montré qu' un stade NYHA III et IV résiduel ( p=0.001, RR 4.55, 95 % IC :1.85 -14.29), une mauvaise fraction d'éjection préopératoire(p=0.013, RR 1.09, 95 % IC 1.02 -1.18), ,une régurgitation mitrale d'origine fonctionnelle (p=0.018, RR 4.17, 95% IC 1.32-16.67) ainsi qu'une étiologie ischémique (p=0.049, RR 3.13, 95% IC 1.01-10.0) constituaient tous des prédicteurs indépendant de mortalité. Une régurgitation mitrale persistante au 7 e jour post-opératoire (p= 0.005, RR 4.55, 95 % IC :1.56 -20.0), un âge inférieur à 60 ans (p = 0.012, RR 8.7, 95 % IC 2.44 - 37.8) et l'absence d'anneau prothétique (p = 0.034, RR 4.76, 95 % IC 1.79-33.3) se sont tous révélés être des facteurs de risque indépendant de réopération. Conclusion: Les réparations mitrales sont accompagnées d'une excellente survie à long terme même si leur évolution peut être influencée négativement par de nombreux facteurs de risques periopératoires. Les risques de réopération sont plus élevés chez des patients jeunes présentant une régurgitation mitrale résiduelle et n'ayant pas bénéficié de la mise en place d'un anneau prothétique.

A cell-based model of extracellular-matrix-guided endothelial cell migration during angiogenesis.

Angiogenesis, the formation of new blood vessels sprouting from existing ones, occurs in several situations like wound healing, tissue remodeling, and near growing tumors. Under hypoxic conditions, tumor cells secrete growth factors, including VEGF. VEGF activates endothelial cells (ECs) in nearby vessels, leading to the migration of ECs out of the vessel and the formation of growing sprouts. A key process in angiogenesis is cellular self-organization, and previous modeling studies have identified mechanisms for producing networks and sprouts. Most theoretical studies of cellular self-organization during angiogenesis have ignored the interactions of ECs with the extra-cellular matrix (ECM), the jelly or hard materials that cells live in. Apart from providing structural support to cells, the ECM may play a key role in the coordination of cellular motility during angiogenesis. For example, by modifying the ECM, ECs can affect the motility of other ECs, long after they have left. Here, we present an explorative study of the cellular self-organization resulting from such ECM-coordinated cell migration. We show that a set of biologically-motivated, cell behavioral rules, including chemotaxis, haptotaxis, haptokinesis, and ECM-guided proliferation suffice for forming sprouts and branching vascular trees.

Perineal stapled prolapse resection for external rectal prolapse: is it worthwhile in the long-term?

BACKGROUND: Perineal stapled prolapse (PSP) resection is a novel operation for treating external rectal prolapse. However, no long-term results have been reported in the literature. This study analyses the long-term recurrence rate, functional outcome, and morbidity associated with PSP resection. METHODS: Nine consecutive patients undergoing PSP resection between 2007 and 2011 were prospectively followed. Surgery was performed by the same surgeons in a standardised technique. Recurrence rate, functional outcome, and complication grade were prospectively assessed. RESULTS: All 9 patients undergoing PSP resection were investigated. The median age was 72 years (range 25-88 years). No intraoperative complications occurred. Faecal incontinence, preoperatively present in 2 patients, worsened postoperatively in one patient (Vaizey 18-22). One patient developed new-onset faecal incontinence (Vaizey 18). The median obstructive defecation syndrome score decreased postoperatively significantly from 11 (median; range 8-13) to 5 (median; range 4-8) (p < 0.005). At a median follow-up of 40 months (range 14-58 months), the prolapse recurrence rate was 44 % (4/9 patients). CONCLUSIONS: The PSP resection is a fast and safe procedure associated with low morbidity. However, the poor long-term functional outcome and the recurrence rate of 44 % warrant a cautious patient selection.

TRPM1 is mutated in patients with autosomal-recessive complete congenital stationary night blindness.

Night vision requires signaling from rod photoreceptors to adjacent bipolar cells in the retina. Mutations in the genes NYX and GRM6, expressed in ON bipolar cells, lead to a disruption of the ON bipolar cell response. This dysfunction is present in patients with complete X-linked and autosomal-recessive congenital stationary night blindness (CSNB) and can be assessed by standard full-field electroretinography (ERG), showing severely reduced rod b-wave amplitude and slightly altered cone responses. Although many cases of complete CSNB (cCSNB) are caused by mutations in NYX and GRM6, in approximately 60% of the patients the gene defect remains unknown. Animal models of human diseases are a good source for candidate genes, and we noted that a cCSNB phenotype present in homozygous Appaloosa horses is associated with downregulation of TRPM1. TRPM1, belonging to the family of transient receptor potential channels, is expressed in ON bipolar cells and therefore qualifies as an excellent candidate. Indeed, mutation analysis of 38 patients with CSNB identified ten unrelated cCSNB patients with 14 different mutations in this gene. The mutation spectrum comprises missense, splice-site, deletion, and nonsense mutations. We propose that the cCSNB phenotype in these patients is due to the absence of functional TRPM1 in retinal ON bipolar cells.

The value of selected in vitro and in silico methods to predict acute oral toxicity in a regulatory context: results from the European Project ACuteTox.

ACuteTox is a project within the 6th European Framework Programme which had as one of its goals to develop, optimise and prevalidate a non-animal testing strategy for predicting human acute oral toxicity. In its last 6 months, a challenging exercise was conducted to assess the predictive capacity of the developed testing strategies and final identification of the most promising ones. Thirty-two chemicals were tested blind in the battery of in vitro and in silico methods selected during the first phase of the project. This paper describes the classification approaches studied: single step procedures and two step tiered testing strategies. In summary, four in vitro testing strategies were proposed as best performing in terms of predictive capacity with respect to the European acute oral toxicity classification. In addition, a heuristic testing strategy is suggested that combines the prediction results gained from the neutral red uptake assay performed in 3T3 cells, with information on neurotoxicity alerts identified by the primary rat brain aggregates test method. Octanol-water partition coefficients and in silico prediction of intestinal absorption and blood-brain barrier passage are also considered. This approach allows to reduce the number of chemicals wrongly predicted as not classified (LD50>2000 mg/kg b.w.).

Unexpected left ventricular free-wall rupture following an aortic catheter-valve implantation.

Our experience with the Sapien trans-apical aortic valve (Edwards Lifesciences Inc., Irvine, CA, USA) has been straightforward without per-procedural mortality except in 1/16 consecutive cases who developed non-apical haemorrhage early after valve implantation. We describe the case of an 84-year-old female carrying a very high operative risk (logistic EuroScore of 44%), who underwent a trans-apical stent-valve implantation for severe and symptomatic aortic valve stenosis (23 mm). Due to massive blood loss, an emergency sternotomy and cannulation for cardiopulmonary bypass resuscitation were necessary to treat (without success) an unusual and unexpected subaortic left ventricular free-wall rupture that occurred few minutes after the stent-valve positioning and implantation. To the best of our knowledge, this is the first described case of a left ventricular free-wall rupture occurring after an otherwise non-complicated standard catheter-based aortic valve replacement.

The ubiquitin-proteasome system in prostate cancer and its transition to castration resistance.

Prostate cancer is the most common carcinoma in the male population. In its initial stage, the disease is androgen-dependent and responds therapeutically to androgen deprivation treatment but it usually progresses after a few years to an androgen-independent phase that is refractory to hormonal manipulations. The proteasome is a multi-unit protease system that regulates the abundance and function of a significant number of cell proteins, and its inhibition results in cancer cell growth inhibition and apoptosis and is already exploited in the clinic with the use of proteasome inhibitor bortezomib in multiple myeloma. In order to be recognized by the proteasome, a target protein needs to be linked to a chain of the small protein ubiquitin. In this paper, we review the role of ubiquitin-proteasome system (UPS) in androgen receptor-dependent transcription as well as in the castration resistant stage of the disease, and we discuss therapeutic opportunities that UPS inhibition offers in prostate cancer.